Immunohistochemical properties of embryonic telocytes in a myogenic microenvironment

Telocytes are a unique interstitial cell type that functions in adulthood and embryogenesis. They have characteristic immunohistochemical phenotypes while acquiring different immunohistochemical properties related to the organ microenvironment. The present study aims to investigate the immunohistochemical features of embryonic telocytes during myogenesis and describe their morphology using light microscopy and TEM. Telocytes represent a major cellular constituent in the interstitial elements. They had distinguished telopodes and podoms and formed a 3D interstitial network in the developing muscles. They formed heterocellular contact with myoblasts and nascent myotubes. Telocytes also had distinctive secretory activity. Telocytes identified by CD34. They also express CD68 and MMP-9 to facilitate the development of new tissues. Expression of CD21 by telocytes may reveal their function in immune defense. They also express VEGF, which regulates angiogenesis. In conclusion, the distribution and immunological properties of telocytes in the myogenic tissue indicate that telocytes provide biological and structural support in the development of the myogenic tissue architecture and organization.


Identification of telocytes in the developing skeletal muscles using semithin sections
Telocytes were identified using toluidine blue (Fig. 2A) and methylene blue (Fig. 2B).They formed a 3D network within the developing muscles.Telocytes were observed around the nascent myotube and myoblast.

Identification of telocytes in the developing muscles using TEM
Telocytes are composed of cell bodies and polymers, which have distinctive podoms.They formed a 3D network surrounding the myoblasts and the nascent myotubes that actively synthesized the myofilaments.Telocytes established direct contact with the myoblasts as well as the myotubes.They release secretory vesicles (Fig. 3).
The number of CD34-positive TCs in the 8-day embryos group (mean ± SE) was 48.00 ± 1.309.The number of VEGF-positive TCs in the 8-day embryos group (mean ± SE) was 40.38 ± 0.5.The number of CD21-positive positive TCs in the 8-day embryos group (mean ± SD) was 37.38 ± 1.34.The number of CD68-positive positive TCs in the 8-day embryos group (mean ± SD) was 37.50 ± 1.512.The results showed that there was a significant difference between the CD34 receptor and other group receptors with (p < 0.05) (Fig. 9).

Discussion
In the current study, telocytes are recognized by their unique podomeres and distinct podoms.Telocytes created a three-dimensional network and established direct contact with both myoblasts and nascent myotubes.These cells are actively synthesizing myofilaments.A previous study indicated that embryonic telocytes are widely   www.nature.com/scientificreports/distributed.They constitute an essential stromal component in the growing skeletal muscle and are arranged into an extensive structure that is dispersed throughout the epimysium, perimysium, and endomysium 13 .In fetal muscle tissue, a reticular network is present which is closely associated with blood vessels, and primary and secondary myotubes.Telocytes networks are more numerous and show higher reactivity to CD34.In contrast, muscle tissue from 12 weeks of gestation shows a significant decrease in the quantity and immunopositivity of Telocytes, where mature myotubes are visible.Between nine and twelve weeks, there are similar changes in the quantity, density, and structure of the muscle stroma.Additionally, blood vessels become most abundant between 10 and 11.5 weeks.According to the authors, Telocytes may be essential throughout the early stages of myogenic development, possibly directing angiogenesis, tissue organization, compartmentalization, and myotube maturation 12 .
In the current investigation, it was found that TCs expressed MMP-9, which indicates proteolytic activity.MMP-9 helps remove extracellular matrix components that may hinder the migratory cells' ability to enter the tissue and creates the new space required for the formation of new structures.MMPs are families of proteolytic enzymes that are essential for tissue remodeling and development as they stimulate the degradation of extracellular matrix components.Gelatinase B, or MMP-9, is a metal-dependent endopeptidase that promotes the growth and migration of cancer cells and is also involved in angiogenesis, as well as the activation of chemokines and cytokines 14 .MMP-9 can proteolytically degrade various ECM components such as collagen types IV, V, XIk' , XIVl' , elastin, aggrecan, link protein, decorin, laminins, entactin, SPARCq, myelin basic protein, 2Mn, 1Pli, IL-1, and proTNF 15 .
In the current study, a certain phagocytic marker was used to identify the properties of embryonic telocytes.The expression of CD68 is unique to phagocytic cells, which have formed endosomal-lysosomal systems.CD68, a  www.nature.com/scientificreports/member of the D-scavenger receptor family, is associated with the maturation of late endosomes and lysosomes.The granules of phagocytes containing CD68 are shown in the illustrations 16 .
The current study reveal that expression of CD68 of telocytes, which linked to phagocytic activities which may help in elimination of unnecessary components may be cellular or debris or degrade matrix components as a result of MMP-9 activity.
TCs express the marker specific for one of the immune cells, CD21.The CD21 endogenous ligand binds to complement component C3 fragments, interferon-alpha, and CD23.CD21 is an integral part of activated B-and T-lymphocytes.An integral part of activated B-and T-lymphocytes is CD21.Interferon, an antiviral cytokine, and DNA-DNA complexes (chromatin) are two innate immune receptors that CD21 interacts with 17 .
Expression of VEGF by TCs reveals the angiogenic role of TCs during development.Angiogenesis is the process by which blood vessels develop from the preexisting vasculature 18 .VEGF expression encourages the development of new blood vessels 19 .VEGF is a member of the platelet-derived growth factor family.VEGF plays several common roles in the development of angiogenesis 20 , vascular permeability 21 , and vascular integrity 22 .The current data revealed that Expression of VEGF development of the myogenic vasculature.
In conclusion, telocytes exhibit proteolytic activity in the extracellular matrix (ECM) by expressing MMP-9.On the other hand, expressing CD68 reveals the phagocytic activity of telocytes.Based on these findings, it can be inferred that telocytes exhibited both proteolytic and phagocytic activity, removing old components and creating new areas for the growth of myogenic tissues.Telocytes also express CD21, which has an essential role in immune function.Expression of VEGF by telocytes promotes the development of new vasculature.The current research indicates that TCs are a distinct cellular component of developing skeletal muscle and may play a role in myogenesis.These unique stromal cells may impact embryonic skeletal muscle tissue architecture and organization through their telopode network and participate in the development of myogenic tissue and angiogenesis.

Ethical approval
The National Ethics Committee of South Valley University and the veterinary authorities in Qena Province, Egypt, authorized the methodology utilized in this study."All procedures were carried out in compliance with the applicable policies and guidelines." The Research Quail Farm, affiliated with the Department of Histology at the Faculty of Veterinary Medicine, South Valley University, Qena, Egypt, is the source of fertilized quail (Coturnix japonica) eggs that we have acquired.The fertilized eggs were incubated at 37.5 °C and 65% relative humidity.The eggs were automatically rotated every six hours following the third day of incubation.Fertilized eggs were removed on the eighth day of incubation and kept for 4 h at − 20 °C before the embryos were removed.Opening the broad end of the eggshells, the embryos that appeared healthy were gently removed.For histochemical and immunohistochemical techniques, three embryos were used, while another three were used for TEM.Prior to fixing, the right and left caudofomralis muscles were carefully extracted.According to previous studies, muscle samples were collected during the early embryonic stages when muscle development and growth takes place 19,23 .

Fixation
Samples used for light microscopic examination was immediately preserved in 10% neutral buffered formalin and immersed in Bouin's solution for half an hour.Subsequently, the fixed samples underwent alcohol and ethanol dehydration at increasing concentrations (70%, 80%, 90%, and 100%).Methyl benzoate was then used to clean the samples.Following that, samples that had been dehydrated were impregnated and embedded in Paraplast (MilliporeSigma, St. Louis, MO, USA).Table 1 lists the paraffin-embedding processing times for the samples.

Histological examination
Serial 5-µm transverse and longitudinal slices were cut with a Leica RM2125 microtome (Leica Microsystems, Wetzlar, Germany).The sections were then maintained at 40 °C in an incubator to ensure they remained dry.The sections were stained for a general histological investigation using hematoxylin and eosin 24 .

Preparations of resin embedding samples for semi-thin sections
The resin-embedding method made use of Karnovsky's fixative.The fixative was prepared in the manner described below 25,26 : 10 mL of each of the following are combined: 30% distilled water, 50% glutaraldehyde, 25% paraformaldehyde, and 50 mL phosphate buffer.Samples from embryos on day eight were used.The neck's skin was carefully peeled off and measured between 2.0 and 3.0 mm in length.Karnovsky fixative, 4 °C overnight (Table 2).
Following osmium tetroxide postfixation, the samples were embedded in resin, washed, and crystallized in a 60 °C oven.Additionally, a pure resin/alcohol mixture was infused into them.The resin was embedded using propylene oxide (Merck, Darmstadt, Germany).A 1:1 mixture of epoxy resin and propylene oxide was then used for around 30 min, and finally, the epoxy resin mix was used for three hours.The epoxy resin composition was prepared by mixing 12 mL of dodecenylsuccinic anhydride (DSAA), 5 mL of Araldite (Huntsman Advanced Materials, The Woodlands, TX, USA), and 5 mL of EMbed 812 (Polysciences Europe GmbH, Eppelheim, Germany).After the samples were embedded, the epoxy resin mixture was heated to 60 °C to polymerize the samples.Next, an accelerator was added to the mixture (2,4,6-Tris[dimethylaminomethyl]phenol; 1.5%).The blocks were incubated at three different temperatures: 60, 70, and 75 °C24,27,28 .Using an ultramicrotome Ultracut E (Reichert-Leica, Germany), semithin sections were cut at 1 μm and stained with methylene blue and toluidine blue 19,23 .Semithin sections were dyed after the resin was dissolved in a saturated alcoholic solution of sodium hydroxide.The stained slices were examined using a Leitz Dialux 20 microscope and a Canon PowerShot A95 digital camera.www.nature.com/scientificreports/ The 5-µm-thick paraffin sections were cleaned for 5 min in a pH 7.4 phosphate-buffered solution (PBS).Subsequently, they underwent xylene dewaxing and were rehydrated using ever-higher ethanol and alcohol grades.The slices were stored in hydrogen peroxide blocks at room temperature to inhibit endogenous peroxidase activity.After that, the parts were given one further 10 mins of running water beneath the faucet.The slides were treated for 20 min at 95-98 °C in a water bath with a 10-mmol sodium citrate buffer (pH 6.0; Table 2) to improve antigen retrieval.After leaving the slides cool for 20 min at room temperature, PBS was used three times for 5 min each time to clean them (pH 7.4).Using Thermo Fisher Scientific's Ultra V Block, nonspecific background staining was blocked for 5 min at room temperature.This was done to avoid staining the artifact by restricting the staining time to no more than 10 min.After the sections were incubated at 4 °C for a whole night with the primary antibody (Table 3) applied, PBS (pH 7.4) was used to wash the sections three times for 5 min each.
The sections were coated with the secondary antibody (Table 3) and left to remain at room temperature for 10 min.Following three 5-min PBS washes (pH 7.4), the slices were left at room temperature for 10 min to be incubated with a streptavidin-peroxidase combination (Thermo Fisher Scientific UK and Lab Vision Corporation).Two milliliters of DAB plus substrate and one drop of DAB plus chromogen were mixed, applied to the sections, and left to remain at room temperature for 5 min to view the bound antibodies.The incubation process was carried out in a humid room.After applying the counterstain, Harris hematoxylin, it was left for 30 s.The sections were dehydrated for 5 min in 100% ethanol twice, after which they were washed in xylene and covered with a DPX (dibutylphthalate polystyrene xylene) mounting solution.We utilized a Leitz Dialux 20 microscope (Leitz GmbH, Oberkochen, Germany) and a Canon PowerShot A95 digital camera (Canon Inc., Tokyo, Japan) to analyze the immunohistochemical staining.

Immunohistochemical procedures for vascular endothelial growth factor (VEGF)
For the two-step immunohistochemical staining process 33, Agilent Technologies, Inc., Santa Clara, California, USA, used the Dako EN Vision + Single Reagent (HRP.Mouse).We applied the staining method that Abdo and associates developed.In summary, paraffin-embedded sections five micrometers thick were dewaxed, rehydrated, and rinsed three times with PBS (pH 7.4), each for 5 min.Methanol was treated with drops of 3% hydrogen peroxide and left to stand at room temperature for 20 min to inhibit endogenous peroxidase activity.After that, it was given a 10-min rinse under running water.To remove antigen, slides were placed in a 10-mm sodium citrate buffer (pH 6.0; Table 2) and heated in a tap water bath for 20 min to a temperature of 95-98 °C.After that, the slides were left to cool at ambient temperature for an additional 20 min.Following that, the sections were washed three times for 5 min each using PBS (pH 7.4).To avoid nonspecific background staining, drops of Dako Protein Block (Agilent Technologies, Inc.) were applied to each segment and allowed to settle at room temperature for 5 min.It should be noted that less staining than anticipated may occur after extended blocking.The sections were subsequently treated with the primary antibody (a distinct type of antibody used in a recent publication that showed reactivity in avian species) 35 .
Table 3 contains a list of all the antibodies used in immunohistochemistry research, along with their names, sources, and working dilutions.Slides were incubated with the secondary antibody for 30 min at room temperature before being washed three times for 5 min each using PBS (pH 7.4) (Table 3).After three 5-min rinses in PBS (pH 7.4), the slides were once again treated with DAB and substrate-chromogen for 5-10 mins at room temperature.This causes a brown precipitate to be produced at the antigen site.Harris hemoxoxylin was used as a counterstain for 30 s on the sections.After being washed in xylene and coated with DPX, the sections were subjected to two rounds of dehydration, lasting 5 min each in 90% and 100% ethanol.As before, we used

Figure 9 .
Figure 9.A column graphic representation of the mean numbers of positive CD34, VEGF, CD68, CD21 receptor TCs at 8 days embryo.Data are presented as the mean ± standard error (n = 8).IHC Expression with different lowercase letters are significantly different (one-way ANOVA: p < 0.05).

Table 2 .
Components of the fixative.

Table 3 .
Identity, sources, and working dilution of antibodies used in Immunohistochemical studies.Antibodies used that showed reactivity in avian species.